The present invention relates to recombinant DNA that encodes the RsaI restriction endonuclease, as well as the RsaI methylase, and to the production of the RsaI restriction endonuclease from the recombinant DNA.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into fragments for molecular cloning and gene characterization.
Restriction endonucleases act by binding to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the DNA molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases recognize and cleave different nucleotide sequences. Over two hundred restriction endonucleases with unique specificities have been identified among thousands of bacterial species that have been examined (Roberts and Macelis, Nucl. Acids Res. 24:223-235, (1996)).
Restriction endonucleases are named according to the bacteria from which they derive. Thus, the bacterium Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5'-TTTAAA-3', 5'-PuGGNCCPy-3' and 5'-CACNNNGTG-3' respectively. Escherichia coli RY13, on the other hand, produces only one restriction enzyme, EcoRI, which recognizes the sequence 5' GAATTC 3'.
Restriction endonucleases usually occur together with one or more companion enzymes termed methyltransferase, the whole forming a restriction-modification (R-M) system. Methyltransferases are complementary to the restriction endonuclease they accompany and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one of the nucleotides within the sequence by the addition of a methyl group to form 5-methylcytosine, N4-methylcytosine, or N6-methyladenine. Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase(s), and therefore it is completely insensitive to the presence of the restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of recombinant DNA technology, it is possible to clone genes and overproduce the enzymes they encode in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e. populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used resistance to bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985)).
A third approach to clone R-M systems is by selection for an active methylase gene (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since R and M genes are usually closely linked, both genes can often be cloned simultaneously by selecting for only one. Selection for the M gene does not always yield a complete restriction system however, but often instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
Another approach is to clone R-M Systems in E.coli by making use of the fact that certain modification genes, when cloned into a new host and adequately expressed, enable the host to tolerate the presence of a different restriction gene (Wilson et al; U.S. Pat. No. 5,246,845).
A more recent method, the "endo-blue method", has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535).
Because purified restriction endonucleases, and to a lesser extent modification methylases, are useful tools for manipulating DNA molecules in the laboratory, there is a commercial incentive to create bacterial strains through recombinant DNA techniques that produce these enzymes in large quantities. Such overexpression strains also simplify the task of enzyme purification.